Antiangiogenic agents increase breast cancer stem cells via the generation of tumor hypoxia.
ABSTRACT: Antiangiogenic therapy has been thought to hold significant potential for the treatment of cancer. However, the efficacy of such treatments, especially in breast cancer patients, has been called into question, as recent clinical trials reveal only limited effectiveness of antiangiogenic agents in prolonging patient survival. New research using preclinical models further suggests that antiangiogenic agents actually increase invasive and metastatic properties of breast cancer cells. We demonstrate that by generating intratumoral hypoxia in human breast cancer xenografts, the antiangiogenic agents sunitinib and bevacizumab increase the population of cancer stem cells. In vitro studies revealed that hypoxia-driven stem/progenitor cell enrichment is primarily mediated by hypoxia-inducible factor 1?. We further show that the Akt/?-catenin cancer stem cell regulatory pathway is activated in breast cancer cells under hypoxic conditions in vitro and in sunitinib-treated mouse xenografts. These studies demonstrate that hypoxia-driven cancer stem cell stimulation limits the effectiveness of antiangiogenic agents, and suggest that to improve patient outcome, these agents might have to be combined with cancer stem cell-targeting drugs.
Project description:Hypoxia induced by antiangiogenic agents is linked to the generation of cancer stem cells (CSCs) and treatment failure through unknown mechanisms. The generation of endothelial cell-independent microcirculation in malignant tumors is defined as tumor cell vasculogenic mimicry (VM). In the present study, we analyzed the effects of an antiangiogenic agent on VM in triple-negative breast cancer (TNBC).Microcirculation patterns were detected in patients with TNBC and non-TNBC. Tientsin Albino 2 (TA2) mice engrafted with mouse TNBC cells and nude mice engrafted with human breast cancer cell lines with TNBC or non-TNBC phenotypes were administered sunitinib and analyzed to determine tumor progression, survival, microcirculation, and oxygen concentration. Further, we evaluated the effects of hypoxia induced with CoCl2 and the expression levels of the transcription factor Twist1, in the presence or absence of a Twist siRNA, on the population of CD133(+) cells and VM in TNBC and non-TNBC cells.VM was detected in 35.8 and 17.8% of patients with TNBC or with non-TNBC, respectively. The growth of tumors in TNBC and non-TNBC-bearing mice was inhibited by sunitinib. The tumors in TA2 mice engrafted with mouse TNBCs and in mice engrafted a human TNBC cell line (MDA-MB-231) regrew after terminating sunitinib administration. However, this effect was not observed in mice engrafted with a non-TNBC tumor cell line. Tumor metastases in sunitinib-treated TA2 mice was accelerated, and the survival of these mice decreased when sunitinib was withdrawn. VM was the major component of the microcirculation in sunitinib-treated mice with TNBC tumors, and the population of CD133+ cells increased in hypoxic areas. Hypoxia also induced MDA-MB-231 cells to express Twist1, and CD133(+) cells present in the MDA-MB-231 cell population induced VM after reoxygenation. Moreover, hypoxia did not induce MDA-MB-231 cells transfected with an sh-Twist1 siRNA cell to form VM and generate CD133(+) cells. Conversely, hypoxia induced MCF-7 cells transfected with Twist to form VM and generate CD133+ cells.Sunitinib induced hypoxia in TNBCs, and Twist1 expression induced by hypoxia accelerated VM by increasing population of CD133(+) cells. VM was responsible for the regrowth of TNBCs sunitinib administration was terminated.
Project description:The effect of antiangiogenic agents targeting the vascular endothelial growth factor A (VEGF-A) pathway has been reported to vary substantially in preclinical studies. The purpose of this study was to investigate the effect of sunitinib treatment on tumor vasculature and oxygenation in melanoma xenografts with different angiogenic profiles. A-07, U-25, D-12, or R-18 melanoma xenografts were grown in dorsal window chambers and given daily treatments of sunitinib (40 mg/kg) or vehicle. Morphologic parameters of tumor vascular networks were assessed from high-resolution transillumination images, and tumor blood supply times (BSTs) were assessed from first-pass imaging movies. Tumor hypoxia was assessed with immunohistochemistry by using pimonidazole as hypoxia marker, and the gene expression and the protein secretion rate of angiogenic factors were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The melanoma lines differed substantially in the expression of VEGF-A, VEGF-C, and platelet-derived growth factor A. Sunitinib treatment reduced vessel densities and induced hypoxia in all melanoma lines, and the magnitude of the effect was associated with the gene expression and protein secretion rate of VEGF-A. Sunitinib treatment also increased vessel segment lengths, reduced the number of small-diameter vessels, and inhibited growth-induced increases in the diameter of surviving vessels but did not change BST. In conclusion, sunitinib treatment did not improve vascular function but reduced vessel density and induced hypoxia in human melanoma xenografts. The magnitude of the treatment-induced effect was associated with the VEGF-A expression of the melanoma lines.
Project description:Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other.
Project description:Carbonic anhydrase IX (CAIX) is a hypoxia regulated metalloenzyme integral to maintaining cellular pH. Increased CAIX expression is associated with poor prognosis in breast cancer. To explore CAIX as a biomarker for breast cancer therapies, we measured plasma CAIX levels in healthy control subjects and in breast cancer patients.In control subjects we evaluated plasma CAIX stability via commercially available ELISA. We then similarly quantified plasma CAIX levels in (1) locally advanced breast cancer (LABC) patients treated with neoadjuvant paclitaxel + sunitinib (T + S) followed by doxorubicin and cyclophosphamide (AC); (2) metastatic breast cancer (MBC) patients treated with systemic chemotherapy.Plasma CAIX levels were stable at room temperature for at least 48 hours in control subjects. Mean baseline plasma CAIX levels were lower in controls compared to patients with LABC or MBC. In LABC, CAIX levels rose significantly in response to administration of antiangiogenic therapy (T + S) (p = 0.02) but not AC (p = 0.37). In patients with MBC treated without an antiangiogenic agent CAIX levels did not change with therapy.Our results suggest that CAIX may be an easily obtained, stable measure of tumor associated hypoxia as well as a useful pharmacodynamic biomarker for antiangiogenic therapy.
Project description:Angiogenesis has become an important target in the treatment of several solid tumors, including breast cancer. As monotherapy, antiangiogenic agents have demonstrated limited activity in metastatic breast cancer (MBC); therefore, they have generally been developed for use in combination with chemotherapies. Thus far, the experience with antiangiogenic agents for MBC has been mixed. The results from one study assessing addition of the monoclonal antibody bevacizumab to paclitaxel led to approval of bevacizumab for MBC. However, the modest improvement of progression-free survival rates in subsequent MBC studies has led to reappraisal of bevacizumab. Phase III studies have not produced evidence supporting use of the multikinase inhibitor sunitinib alone or in combination with MBC chemotherapy. Experience with sorafenib in a phase IIb program indicates potential when used in select combinations, particularly with capecitabine; however, phase III confirmatory data are needed. Although antiangiogenic therapies combined with chemotherapy have increased progression-free survival rates for patients with MBC, increases in overall survival times have not been observed. Some studies have tried to combine antiangiogenic agents such as bevacizumab and sunitinib or sorafenib, but that approach has been limited because of toxicity concerns. Sequential use of antiangiogenic agents with differing mechanisms of action may be an effective approach. Despite setbacks, angiogenesis will likely remain an important target of treatment for selected patients with MBC.
Project description:Gemcitabine (Gem) has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). The present study investigated combinations of gemcitabine with antiangiogenic agents of various mechanisms for PDAC, including bevacizumab (Bev), sunitinib (Su) and EMAP II. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo experiments were performed via murine xenografts. Inhibition of in vitro proliferation of AsPC-1 PDAC cells by gemcitabine (10 µM), bevacizumab (1 mg/ml), sunitinib (10 µM) and EMAP (10 µM) was 35, 22, 81 and 6 percent; combination of gemcitabine with bevacizumab, sunitinib or EMAP had no additive effects. In endothelial HUVECs, gemcitabine, bevacizumab, sunitinib and EMAP caused 70, 41, 86 and 67 percent inhibition, while combination of gemcitabine with bevacizumab, sunitinib or EMAP had additive effects. In WI-38 fibroblasts, gemcitabine, bevacizumab, sunitinib and EMAP caused 79, 58, 80 and 29 percent inhibition, with additive effects in combination as well. Net in vivo tumor growth inhibition in gemcitabine, bevacizumab, sunitinib and EMAP monotherapy was 43, 38, 94 and 46 percent; dual combinations of Gem+Bev, Gem+Su and Gem+EMAP led to 69, 99 and 64 percent inhibition. Combinations of more than one antiangiogenic agent with gemcitabine were generally more effective but not superior to Gem+Su. Intratumoral proliferation, apoptosis and microvessel density findings correlated with tumor growth inhibition data. Median animal survival was increased by gemcitabine (26 days) but not by bevacizumab, sunitinib or EMAP monotherapy compared to controls (19 days). Gemcitabine combinations with bevacizumab, sunitinib or EMAP improved survival to similar extent (36 or 37 days). Combinations of gemcitabine with Bev+EMAP (43 days) or with Bev+Su+EMAP (46 days) led to the maximum survival benefit observed. Combination of antiangiogenic agents improves gemcitabine response, with sunitinib inducing the strongest effect. These findings demonstrate advantages of combining multi-targeting agents with standard gemcitabine therapy for PDAC.
Project description:Using a cell-based reporter gene assay, we screened a library of drugs in clinical use and identified the anthracycline chemotherapeutic agents doxorubicin and daunorubicin as potent inhibitors of hypoxia-inducible factor 1 (HIF-1)-mediated gene transcription. These drugs inhibited HIF-1 by blocking its binding to DNA. Daily administration of doxorubicin or daunorubicin potently inhibited the transcription of a HIF-1-dependent reporter gene as well as endogenous HIF-1 target genes encoding vascular endothelial growth factor, stromal-derived factor 1, and stem cell factor in tumor xenografts. CXCR4(+)/sca1(+), VEGFR2(+)/CD34(+), and VEGFR2(+)/CD117(+) bone-marrow derived cells were increased in the peripheral blood of SCID mice bearing prostate cancer xenografts but not in tumor-bearing mice treated for 5 days with doxorubicin or daunorubicin, which dramatically reduced tumor vascularization. These results provide a molecular basis for the antiangiogenic effect of anthracycline therapy and have important implications for refining the use of these drugs to treat human cancer more effectively.
Project description:Different mechanisms of angiogenesis and vasculogenesis are involved in the development of the tumor vasculature. Among them, cancer stem cells are known to contribute to tumor vasculogenesis through their direct endothelial differentiation. Here, we investigated the effect of anti-angiogenic therapy on vasculogenesis of cancer stem cells derived from breast and renal carcinomas. We found that all the anti-angiogenic approaches impaired proliferation and survival of cancer stem cells once differentiated into endothelial cells in vitro and reduced murine angiogenesis in vivo. At variance, only VEGF-receptor inhibition using the non-specific tyrosine kinase inhibitor Sunitinib or the anti-VEGF-receptor 2 neutralizing antibody, but not VEGF blockade using Bevacizumab, impaired the process of endothelial differentiation in vitro, suggesting a VEGF-independent mechanism. In addition, tyrosine kinase inhibition by Sunitinib but not VEGF blockade using the soluble VEGF trap sFlk1 inhibited the cancer stem cell-induced vasculogenesis in vivo. Accordingly, Sunitinib but not Bevacizumab inhibited the induction of hypoxia-inducible factor pathway occurring during endothelial differentiation under hypoxia. The present results highlight a differential effect of VEGF-receptor blockade versus VEGF inhibition in tumor vascularization. VEGFR blockade inhibits the process of tumor vasculogenesis occurring during tumor hypoxia whereas the effect of VEGF inhibition appears restricted to differentiated endothelial cells.
Project description:Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.